US7977366B2 - Treating an inflammatory disorder or inhibiting respiratory burst in adherent neutrophils with chemical inhibitors of neutrophil activation - Google Patents

Treating an inflammatory disorder or inhibiting respiratory burst in adherent neutrophils with chemical inhibitors of neutrophil activation Download PDF

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US7977366B2
US7977366B2 US10/575,683 US57568306A US7977366B2 US 7977366 B2 US7977366 B2 US 7977366B2 US 57568306 A US57568306 A US 57568306A US 7977366 B2 US7977366 B2 US 7977366B2
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neutrophils
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Hyunsil Han
Gang Lin
Carl Nathan
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Cornell Research Foundation Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/14—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D231/18—One oxygen or sulfur atom
    • C07D231/20—One oxygen atom attached in position 3 or 5
    • C07D231/22—One oxygen atom attached in position 3 or 5 with aryl radicals attached to ring nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/62—Benzothiazoles
    • C07D277/68—Benzothiazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
    • C07D277/70—Sulfur atoms
    • C07D277/74—Sulfur atoms substituted by carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Definitions

  • the present invention relates to methods of treating inflammatory disorders and inhibiting soluble effector-triggered respiratory burst triggered by bacteria in adherent neutrophils without inhibiting neutrophil degranulation in response to either soluble effectors or bacteria.
  • Neutrophils are polymorphonuclear leukocytes (PMNs) of the blood that play a major role in almost all forms of acute inflammation and many forms of chronic inflammation, such as rheumatoid arthritis. When triggered, neutrophils secrete potent oxidants and proteases that contribute to inflammation but also help protect the host from infection. Neutrophils respond to soluble inflammatory mediators by migrating to the site of tissue injury and by ingesting and destroying invading pathogens and damaged tissue, leading, ultimately, to resolution and tissue repair. An inherent dilemma of anti-inflammatory therapy, the risk of impairing host defense, is particularly problematic with neutrophils.
  • neutrophils To participate effectively in the inflammatory process, neutrophils must leave the bloodstream and migrate into the tissues. The initial step in this process is adherence to the vascular endothelium. Adherence of neutrophils to endothelium, their diapedesis from venules into tissues, and their release of peptides, proteases, and reactive oxygen intermediates underlie both their killing of bacteria and their damage to tissues.
  • TNF tumor necrosis factor
  • MIP-1 macrophage inflammatory protein
  • G-CSF granulocyte-specific colony stimulating factor
  • GM-CSF granulocyte-macrophage-specific colony stimulating factor
  • TNF has been studied extensively with respect to both the mechanisms by which it activates adherent neutrophils (Nathan C. F., “Neutrophil Activation on Biological Surfaces. Massive Secretion of Hydrogen Peroxide in Response to Products of Macrophages and Lymphocytes,” J. Clin. Invest. 80:1550-1560 (1987); De La Harpe et al., “Adenosine Regulates the Respiratory Burst of Cytokine-Triggered Human Neutrophils Adherent to Biologic Surfaces,” J. Immunol.
  • neutrophils sense and respond to gradients of activating agents (chemokines), which stimulate the neutrophil to move towards the gradient in a process known as chemotaxis (Brown S S., “Structure and Function of Profilin,” Cell Motil Cytoskel 17:71-75 (1990); Southwick et al., “Contractile Proteins in Leukocyte Function,” Semin Hematol 30:305-310(1984)).
  • chemokines activating agents
  • Activated neutrophils are also capable of phagocytosing, i.e., engulfing foreign or damaged material, an important aspect of the inflammatory response.
  • neutrophils extend pseudopodia, which engulf the offending material, trapping the material inside the cell in a compartment known is a phagosome (Wright S. D., “Receptors for Complement and the Biology of Phagocytosis,” In Inflammation 2nd ed. 477-496 Raven Press New York (1992)). Cytoplasmic-bound granules, the primary and secondary granules of the neutrophil, which contain a multitude of effectors proteins, fuse with the phagosome, placing effector proteins in direct contact with the ingested material.
  • Components of the primary granules include lysozyme, which can digest the peptidoglycan component of most bacterial cell walls, and elastase, cathepsin G, defensins, bacterial permeability-increasing protein (BPI), and myeloperoxidase, which converts hydrogen peroxide generated by NADPH oxidase and hydrochloric acid to hypochlorous acid, all with inherent antibacterial activity.
  • proteins contained in the secondary granules are lactoferrin, an iron-binding protein with some antibacterial activity.
  • the secondary granules also contain stored sources of CR3 and other receptors for neutrophil activation agents, as well as stored membrane components of NADPH oxidase.
  • NADPH oxidase is a crucial component of the neutrophil host defense mechanism. This enzyme assembles on the phagosomal membrane to generate superoxide anion from molecular oxygen and free electrons. Superoxide is then converted to the toxic metabolite hydrogen peroxide by the actions of superoxide dismutase, or to hypochlorous acid by the primary granule component myeloperoxidase (DeLeo et al., “Assembly of Phagocyte NADPH Oxidase: Molecular Interaction of Oxidase Proteins,” J.
  • Leukocyte Biol 60:677-691 (1996); Leusen et al., “Interactions Between the Components of the Human NADPH Oxidases: Intrigues in the Phox Family,” J. Clin Lab Med 128: 461-476 (1996); Wientjes et al., NADPH Oxidase and the Respiratory Burst,” Semin Cell Biol 6:357-365 (1995); Henderson et al., “NADPH Oxidases of Neutrophils,” Biochim Biophys Acta 1273:87-107 (1996)). This ability to generate toxic oxygen metabolites is crucial to host defense against microbes.
  • the histotoxic impact of neutrophils is prominent in several inflammatory settings that are not thought to involve bacterial infection, such as rheumatoid arthritis, Crohn's Disease, and ischemia-perfusion syndrome, or in which neutrophil-mediated injury can occur at sites remote from invading bacteria, as in the acute respiratory distress and systemic inflammatory response syndromes.
  • the stimuli that activate neutrophils in these settings are host-derived mediators (e.g., TNF), rather than bacteria themselves.
  • TNF host-derived mediators
  • What is needed is a method for inhibiting some inflammatory functions of activated neutrophils while sparing antimicrobial functions. Such a method would be particularly useful for preventing and treating inflammatory disorders related to respiratory burst in neutrophils mediated by effector proteins such as TNF.
  • the present invention is directed to overcoming these and other deficiencies in the art.
  • the present invention relates to a method of treating an inflammatory disorder in a subject.
  • This method involves administering to a subject an effective amount of a compound selected from the group including the compound having the formula:
  • R 1 is selected from the group consisting of: H, substituted or unsubstituted phenyl, and substituted or unsubstituted naphthalene
  • R 2 is selected from the group consisting of: H, C 1 -C 4 alkyl, substituted or unsubstituted phenyl, NH 2 , CH 2 COOCH 2 CH 3 , and COOCH 2 CH 3 .
  • Another compound for use in this method is a compound having the formula:
  • R 1 is selected from the group consisting of: H and C 1 -C 4 alkyl
  • R 2 is selected from the group consisting of: C 1 -C 4 alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted furan, substituted or unsubstituted cyclohexyl, substituted or unsubstituted naphthalene, substituted or unsubstituted indol, substituted or unsubstituted pyridine, and substituted or unsubstituted thiophene.
  • Another compound for use in this method is a compound having the formula:
  • R 1 is selected from the group consisting of: H and C 1 -C 4 alkyl
  • R 2 is selected from the group consisting of: substituted or unsubstituted pyridine and substituted or unsubstituted phenyl.
  • Yet another compound for use in this method is a compound having the formula:
  • R 1 is substituted or unsubstituted phenyl
  • R 2 is C 1 -C 4 alkyl
  • R 3 is a substituted or unsubstituted quinoline, with or without a linking group.
  • Still another compound for use in this method of the present invention is a compound having the formula:
  • R 1 is substituted or unsubstituted phenyl
  • R 2 is C 1 -C 4 alkyl
  • R 3 is substituted or unsubstituted benzoylhydrazino.
  • the present invention also relates to a method of inhibiting respiratory burst in adherent neutrophils without inhibiting neutrophil degranulation in or bacterial killing by neutrophils.
  • This method involves contacting adherent neutrophils with an effective amount a chemical compound selected from the group including a compound having the formula:
  • R 1 is selected from the group consisting of: H, substituted or unsubstituted phenyl, and substituted or unsubstituted naphthalene
  • R 2 is selected from the group consisting of: H, C 1 -C 4 alkyl, substituted or unsubstituted phenyl, NH 2 , CH 2 COOCH 2 CH 3 , and COOCH 2 CH 3 .
  • Another compound for use in this method is a compound having the formula:
  • R 1 is selected from the group consisting of: H and C 1 -C 4 alkyl
  • R 2 is selected from the group consisting of: C 1 -C 4 alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted furan, substituted or unsubstituted cyclohexyl, substituted or unsubstituted naphthalene, substituted or unsubstituted indol, substituted or unsubstituted pyridine, and substituted or unsubstituted thiophene.
  • Another compound for use in this method is a compound having the formula:
  • R 1 is selected from the group consisting of: H and C 1 -C 4 alkyl
  • R 2 is selected from the group consisting of: substituted or unsubstituted pyridine, and substituted or unsubstituted phenyl.
  • Yet another compound for use in this method is a compound having the formula:
  • R 1 is substituted or unsubstituted phenyl
  • R 2 is C 1 -C 4 alkyl
  • R 3 is selected from the group consisting of substituted or unsubstituted quinoline, with or without a linking group.
  • Still another compound for use in this method of the present invention is a compound having the formula:
  • R 1 is substituted or unsubstituted phenyl
  • R 2 is C 1 -C 4 alkyl
  • R 3 is substituted or unsubstituted benzoylhydrazino.
  • the present invention provides a method of selectively inhibiting the immune and inflammatory responses triggered in neutrophils by cellular mediators, thereby preventing or treating an inflammatory disorder in a subject without compromising the subject's ability to stage an immune response against foreign microbes.
  • FIG. 1 shows the combinatorial chemistry scheme for screening 15,000 compounds for inhibition of neutrophil respiratory burst.
  • FIG. 2 is a diagram showing the TNF-inhibition assay used for initial screening of compounds from the combinatorial library.
  • FIG. 3 is a diagram of TNF-PMA counter-screening of potentially useful compounds from the combinatorial library.
  • FIG. 4 shows some of the compounds found to be active or inactive during the screening process.
  • FIGS. 5A-F show several small chemical compounds that separately control neutrophil respiratory burst and degranulation. Neutrophils were incubated with the indicated concentrations of each compound for 37° C. for 30 min before stimulation with TNF (100 ng/ml, ⁇ ). H 2 O 2 release measured at 90 min is displayed as % H 2 O 2 release of TNF or PMA alone, in the absence of any compound.
  • FIG. 5A shows the effect of Comp I (23G01) on neutrophil regulatory burst;
  • FIG. 5B shows the effect of Comp II (53F03);
  • FIG. 5C shows the effect of Comp II′ (9G01);
  • FIG. 5D shows the effect of Comp III (55D01);
  • FIG. 5E shows the effect of Comp IV (109G01), and
  • FIG. 5F shows the effect of control Comp V (49G04).
  • FIGS. 6A-D show the effect of other small compounds of the present invention on TNF and PMA triggered neutrophil respiratory burst and degranulation. The experiments were carried out essentially as described for FIGS. 5A-F .
  • FIGS. 6A-D show the effect of compounds 11C09, 11D09, 11G02, and 12D02, respectively, on TNF and PMA respiratory burst.
  • FIGS. 7A-C show the effect of still more small molecule compounds of the present invention on TNF and PMA triggered neutrophil respiratory burst and degranulation. The experiments were carried out essentially as described for FIGS. 5A-F .
  • FIGS. 7A-C show, respectively, the effect of compounds 10E02, 10G04, and 11B09.
  • FIG. 8 shows the reversibility of the inhibition of TNF triggered respiratory burst by compounds of the present invention.
  • Neutrophils incubated with DMSO (D) or each compound (I-V) were washed (hatched bar) or not (black solid bar) with cold KRPG and plated in 96 well plates before stimulation with TNF (100 ng/ml) or an equivalent volume of KRPG buffer as control.
  • TNF 100 ng/ml
  • Results are expressed as means ⁇ SEM for triplicates.
  • FIGS. 9A-B shows the effect of N-formyl-methionyl-leucyl-phenylalanine (fMLF) stimulated respiratory burst when neutrophils are treated with compounds of the present invention.
  • FIG. 9A shows respiratory burst of neutrophils incubated with DMSO (D), Comp II (53F03), Comp V (49G04)(V), or a buffer control (NS) for 30 min and stimulated with fMLF(100 nM).
  • D DMSO
  • Comp II 53F03
  • Comp V 49G04
  • NS buffer control
  • FIG. 9B shows respiratory burst of neutrophils incubated with DMSO (D), Comp I (23G01)(I), Comp III (55D01)(III), Comp IV (109G01)(IV), and Comp V (49G04)(V), or buffer control (NS) for 30 min and stimulated with fMLF(100 nM). H 2 O 2 release is expressed as means ⁇ SEM for triplicates.
  • FIGS. 10A-B shows the results of bacteria triggered respiratory burst when neutrophils are treated with the small molecule compounds of the present invention prior to triggering respiratory burst by contact with bacteria.
  • Neutrophils were incubated with either DMSO (D), Comp I-V, or a buffer control (NS) for 30 min and stimulated with L. monocytogenes or S. typhimurium .
  • FIG. 10A shows the results using Comp II as active molecule and Comp V as inactive molecule.
  • FIG. 10B shows inhibition by Comp I, III, and IV, compared to Comp V and DMSO controls. H 2 O 2 release is expressed as means ⁇ SEM for triplicates.
  • FIGS. 11A-B show the influence of Comp I-V (as described above) on the TNF triggered degranulation of neutrophils.
  • Neutrophils were incubated with TNF, and cell supernatants were assayed for the presence of lactoferrin (LF), in FIG. 11A , and myeloperoxidase (MPO), FIG. 11B .
  • LF lactoferrin
  • MPO myeloperoxidase
  • FIGS. 12A-B are photographs demonstrating the influence of Comp I-V on neutrophil spreading.
  • Neutrophils were plated on FBS-coated glass coverslips and incubated with DMSO (D), a compound of the present invention, or a buffer (NS) at 37° C. for 30 min before stimulation with TNF (100 ng/ml), PMA (100 ng/ml), or an equal volume of KRPG, fixed, and photographed with phase-contrast microscope.
  • FIG. 12 A shows results with Comp II (53F03) and Comp V (49G04).
  • FIG. 12B shows results with Comp I, III, IV, and V.
  • FIGS. 13A-B show the results of the transmigration of neutrophils through a TNF activated human umbilical vein endothelial cell (HUVEC) layer.
  • Neutrophils were incubated with or without a compound of the present invention compound at RT for 30 min, layered on non-stimulated (grey bar) or TNF (50 pg/ml, black bar) stimulated HUVECs, and allowed to transmigrate at 37° C. for 30 min. Unbound cells were washed off with phosphate-buffered saline (PBS) and fixed. Cells on top of and below the HUVEC layers were stained and counted under the microscope. Results of the experiment with Comp II are shown.
  • PBS phosphate-buffered saline
  • FIGS. 14A-D show the impact of Comp I-V on several known tyrosine kinases.
  • FIG. 14A shows the effect of Comp I-V on TNF triggered tyrosine phosphorylation of total cellular proteins. Neutrophil lysates, prepared as indicated in the examples, were separated by SDS-PAGE and western-blotted (WB) with anti-phosphotyrosine antibody.
  • FIG. 14B shows the effect of Comp I-IV on TNF triggered Src activity. Recombinant Src kinase was incubated with each compound and assayed for its kinase activity in vitro.
  • FIG. 14C shows the effect of Comp I-V on TNF triggered Syk activity.
  • FIG. 14D shows the activity of recombinant Syk pre-incubated with Comp I, II, III, IV or V, DMSO (D) or and measured in vitro.
  • FIG. 15 shows the effect of TNF-induced phosphorylation of endogenous Pyk2.
  • Neutrophils were treated with Comp I-V, and cell lysates prepared as indicated were western blotted (WB) with phosphorylated Pyk2 specific antibody.
  • FIG. 16 is a schematic diagram of the phagocyte oxidase/NADPH oxidase (Phox) pathway.
  • FIG. 17 shows the translocation of phox 47 subunit from the cytosol to the cell membrane.
  • Neutrophils were incubated with DMSO or Comp II at 37° C. for 30 min and stimulated with TNF (T), PMA (P), or a buffer alone as a control (NS). After stimulation, membrane fractions were collected through ultracentrifugation, separated by SDS-PAGE and western-blotted (WB) with anti-phox 47 antibody.
  • the present invention relates to a method of treating an inflammatory disorder in a subject.
  • This method involves administering to a subject an effective amount of a compound under conditions to treat the inflammatory disorder.
  • One suitable compound for use in carrying out this method is a compound having the general formula:
  • R 1 is H, substituted or unsubstituted phenyl, or substituted or unsubstituted naphthalene
  • R 2 is H, C 1 -C 4 alkyl, substituted or unsubstituted phenyl, NH 2 , CH 2 COOCH 2 CH 3 , or COOCH 2 CH 3 .
  • 53F03 also called Comp I herein
  • 53F03 has the formula:
  • Another suitable compound for use in this method has the general formula:
  • R 1 is H or C 1 -C 4 alkyl
  • R 2 is C 1 -C 4 alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted furan, substituted or unsubstituted cyclohexyl, substituted or unsubstituted naphthalene, substituted or unsubstituted indol, substituted or unsubstituted pyridine, or substituted or unsubstituted thiophene.
  • This compound called hereinafter 109G01, has the formula:
  • R 1 is H or C 1 -C 4 alkyl
  • R 2 is substituted or unsubstituted pyridine or substituted or unsubstituted phenyl.
  • this compound called 55D01 hereinafter, has the formula:
  • R 1 is substituted or unsubstituted phenyl
  • R 2 is C 1 -C 4 alkyl
  • R 3 is substituted or unsubstituted quinoline, with or without a linking group.
  • 9G01 also called Comp II'
  • Still another suitable compound for use in this method of the present invention is a compound having the general formula:
  • R 1 is substituted or unsubstituted phenyl
  • R 2 is C 1 -C 4 alkyl
  • R 3 is substituted or unsubstituted benzoylhydrazino.
  • Exemplary compounds having this general formula as a base include, without limitation, the compound called hereinafter 10E02, having the formula:
  • a second suitable compound of this aspect of the present invention having the same generic base formula as 10E02 is the compound called 10G04 hereinafter, having the following formula:
  • the compounds are administered to a subject under conditions effective to treat the inflammatory disorder.
  • the compounds used according to the methods of the present invention can be administered alone or as a pharmaceutical composition, which includes the compound(s) and a pharmaceutically-acceptable carrier.
  • the compounds of the present invention are typically provided as a pharmaceutical composition.
  • the pharmaceutical composition can also include suitable excipients, or stabilizers, and can be in solid or liquid form such as tablets, capsules, powders, solutions, suspensions, or emulsions.
  • the composition will contain from about 0.01 to 99 percent, preferably from about 5 to 95 percent of active compound(s), together with the carrier.
  • the compounds of the present invention when combined with pharmaceutically or physiologically acceptable carriers, excipients, or stabilizers, whether in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions, can be administered orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes, for example, that of the nose, throat, and bronchial tubes including, for example, by inhalation.
  • the compounds can be administered orally as a solid or as a solution or suspension in liquid form, via injection as a solution or suspension in liquid form, or via inhalation of a nebulized solution or suspension.
  • the solid unit dosage forms can be of the conventional type.
  • the solid form can be a capsule, such as an ordinary gelatin type containing the compounds of the present invention and a carrier, for example, lubricants and inert fillers such as, lactose, sucrose, or cornstarch.
  • these compounds are tableted with conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, or gelatin, disintegrating agents, such as cornstarch, potato starch, or alginic acid, and a lubricant, like stearic acid or magnesium stearate.
  • conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, or gelatin
  • disintegrating agents such as cornstarch, potato starch, or alginic acid
  • a lubricant like stearic acid or magnesium stearate.
  • solutions or suspensions of these materials can be prepared in a physiologically acceptable diluent with a pharmaceutical carrier.
  • a pharmaceutical carrier include sterile liquids, such as water and oils, with or without the addition of a surfactant and other pharmaceutically and physiologically acceptable carrier, including adjuvants, excipients or stabilizers.
  • sterile liquids such as water and oils
  • surfactant and other pharmaceutically and physiologically acceptable carrier including adjuvants, excipients or stabilizers.
  • Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil.
  • water, saline, aqueous dextrose, and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.
  • the compound in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • the materials of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.
  • the compound is present in a carrier which forms a composition in the form of a cream, lotion, solution, and/or emulsion.
  • the composition can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.
  • Suitable disorders to be treated or prevented in all aspects of the present invention present herein above or below are disorders in which a major pathogenic role is assigned to inflammation, including, without limitation, ischemia-reperfusion injury (occlusive and embolic stroke and myocardial infarction, type I diabetes mellitus, asthma, chronic obstructive pulmonary disease, gout, pre-term labor, sarcoidosis, ulcerative colitis, rheumatoid arthritis, osteoarthritis, xenograft rejection, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, pemphigus, chronic obstructive pulmonary disease, systemic lupus erythematosus, atopic dermatitis, vasculitides (Wegener's Syndrome, Goodpasture's Syndrome, giant cell arteritis, polyarteritis nodosa), multiple sclerosis, Alzheimer's Disease, and Crohn's Disease (regional enter
  • the method of the present invention is also useful for treating diseases of infectious origin in which inflammation may contribute as much to pathology as does microbial toxicity, including, without limitation, sepsis syndrome, post-streptococcal glomerulonephritis, hepatitis C, Neisserial or Pneumococcal meningitis, Helicobacter pylori gastritis, influenza virus pneumonia, tuberculosis, leprosy (tuberculoid form), filariasis, cystic fibrosis, bacterial dysentery, and Chagas Disease ( Trypanosoma cruzi ).
  • Additional conditions or disorders encompassed by the methods of the present invention are diseases of diverse origin in which post-inflammatory fibrosis is a major cause of pathology. These diseases include, without limitation: schistosomiasis, idiopathic pulmonary fibrosis, hepatic cirrhosis (post-viral or alcoholic), radiation-induced pulmonary fibrosis, chronic allograft rejection, and bleomycin-induced pulmonary fibrosis.
  • Another aspect of the present invention is a method of inhibiting respiratory burst in adherent neutrophils without inhibiting neutrophil degranulation in or bacterial killing by the neutrophils.
  • This method involves contacting adherent neutrophils with an effective amount of the above listed chemical compounds. This involves the formulations and modes of administration described above.
  • This aspect of the present invention may be carried out by contacting adherent neutrophils in vitro using methods known in the art, including, but not limited to, adding the compounds described herein above to adherent neutrophils in a suitable cell culture system. This aspect of the present invention may also be carried out by contacting neutrophils in vivo as described above.
  • the respiratory burst is triggered by an protein effector agent such as a chemokine, a cytokine, a complement component, a bacterium, or a bacterial factor.
  • an protein effector agent such as a chemokine, a cytokine, a complement component, a bacterium, or a bacterial factor.
  • Suitable chemokines for this aspect of the present invention include, without limitation, macrophage inflammatory protein-1 (MIP-1) and interleukin-8 (IL-8), as well as the chemoattractant complement component C5a.
  • MIP-1 macrophage inflammatory protein-1
  • IL-8 interleukin-8
  • Suitable cytokines for this aspect of the present invention include, without limitation, tumor necrosis factor (TNF), lymphotoxin, granulocyte-specific colony stimulating factor (G-CSF), and granulocyte/macrophage-specific colony stimulating factor (GM-CSF).
  • TNF tumor necrosis factor
  • G-CSF granulocyte-specific colony stimulating factor
  • GM-CSF granulocyte/macrophage-specific colony stimulating factor
  • bacteria suitable as a trigger of respiratory burst in this aspect of the present invention are bacteria. This includes, without limitation, whole bacteria, bacterial cell wall components, and secreted or shed bacterial products.
  • Suitable bacteria of this aspect of the present invention include, without limitation, the Gram-negative bacterium Salmonella enterica Var. typhimurium and the Gram-positive bacterium Listeria monocytogenes .
  • Suitable secreted or shed bacterial products of this aspect of the present invention include, without limitation, amino-terminally formylated peptides such as N-formyl-methionyl-leucyl-phenylalanine (fMLF).
  • High Throughput Screening of a chemical compound library selected by Dr. Tarun Kapoor at Rockefeller University was carried out to identify specific inhibitors of TNF and PMA-triggered H 2 O 2 release by primary human neutrophils.
  • the library used for screening consists of 15,000 compounds generated from 125 combinatorial templates each template with 200 different side chain modifications.
  • the compounds are “drug like” compounds, i.e., small molecules that have high membrane permeability for efficient delivery of molecules across the cell membrane.
  • 460 compounds showed over 90% inhibition at 20 ⁇ M during primary screening.
  • Neutrophils were isolated to >95% purity from heparinized (10 U/ml) blood of normal human donors using PolymorphprepTM (Axis-Shield PoC AS, Norway) according to the manufacturer's instructions. Contaminating erythrocytes were lysed by hypotonic shock for 45 seconds with 0.2% saline. Neutrophils were resuspended in Krebs-Ringer phosphate with glucose (KRPG) formulated as described (De la Harpe et al., “A Semi-Automated Micro-Assay for H2O2 Release by Human Blood Monocytes and Mouse Peritoneal Macrophages,” J Immunol Methods 78:323-336 (1985), which is hereby incorporated by reference in its entirety).
  • KRPG Krebs-Ringer phosphate with glucose
  • FIGS. 2-3 demonstrate the screening scheme that identified the compounds of the present invention. Briefly, 15,000 compounds were screened to identify specific inhibitors of TNF-triggered H 2 O 2 release in human neutrophils. H 2 O 2 release was measured as above except a few modifications to adapt to high throughput screening. Black, instead of clear, 96-well tissue culture plates (Falcon, Cat. No. 353945, Fisher Scientific, Hampton, NH) were used to reduce the background fluorescence reading.
  • Perkin-Elmer Fusion microplate reader was used for recording fluorescence, Titertek Multidrop 96/384 for bulk reagent dispensing, Bi-Tek Elx 405 Select Plate Wash system for plate washing, and Perkin Elmer MiniTrakV liquid handling robot (Perkin Elmer, Foster City, Calif.) for the delivery of compound aliquots to each well. Percent inhibition by each compound was calculated using the formula:
  • T0 and T90 are fluorescence readings at each well at 0 and 90 min, respectively.
  • Tc0 and Tc90 are the mean fluorescence readings in the compound free control wells at 0 and 90 min, respectively.
  • Compounds that showed over 90% inhibition on TNF triggered respiratory burst were retested on both TNF and PMA triggered burst to eliminate non-specific, toxic chemicals or PKC inhibitors.
  • Glass coverslips were placed in 12 well tissue culture plate and coated with FBS in 5% CO 2 at 37° C. for at least 1 h and washed three times with 0.9% saline.
  • Neutrophils (2 ⁇ 10 6 ) were added to each well containing a FBS-coated glass coverslip and 1 ml of reaction mixture and incubated or not with each compound at 37° C. for 30 min before stimulation with TNF (100 ng/ml), PMA (100 ng/ml) or an equal volume of KRPG.
  • TNF 100 ng/ml
  • PMA 100 ng/ml
  • Cells were fixed with 2% paraformaldehyde and 3.7% formaldehyde buffer, and photographed with phase-contrast microscope.
  • Respiratory burst was measured as described elsewhere (De la Harpe et al., “A Semi-Automated Micro-Assay for H2O2 Release by Human Blood Monocytes and Mouse Peritoneal Macrophages,” J Immunol Methods 78:323-336 (1985), which is hereby incorporated by reference in its entirety). Briefly, 96-well flat-bottomed plates (Primaria, Falcon) were coated with 50 ⁇ l/well of FBS in 5% CO 2 at 37° C. for at least 1 h and washed three times with 0.9% saline.
  • Neutrophils (1.5 ⁇ 10 4 ) were added to triplicate wells containing 100 ⁇ l of reaction mixture (2.4 nm scopoletin, 0.5 ⁇ g horseradish peroxidase (HRP), and 1 mM NaN 3 ) and stimulated with either buffer control, TNF (PreproTech, London, UK) or phorbol myristate acetate (PMA), each at 100 ng/ml.
  • reaction mixture 2.4 nm scopoletin, 0.5 ⁇ g horseradish peroxidase (HRP), and 1 mM NaN 3
  • TNF PreproTech, London, UK
  • PMA phorbol myristate acetate
  • FIGS. 5A-F The chemical structures of the five specific compounds are illustrated in FIGS. 5A-F .
  • IC 50 of Comp I is about 0.18 ⁇ 0.04 ⁇ M, 1.6 ⁇ 0.4 ⁇ M for Comp II, 22 nm for Comp II′, and 2.5 ⁇ 1.5 ⁇ M for Comp III.
  • Comp IV displayed very variable IC 50 ranging from 3 nM-2 ⁇ M.
  • FIGS. 6A-D show the on TNF and PMA triggered H 2 O 2 release using compounds 11C09, 11D09, 11G02, and 12D02, respectively, and FIGS. 7A-C show the effect of compounds 10E2, 10G04, and 11B09, respectively.
  • Neutrophils were incubated with test compounds I-V or DMSO at 37° C. for 30 min in FBS coated tubes (Falcon, Cat. Number 352063), washed twice with cold KRPG, and plated in 96 well plates before stimulation with TNF (100 ng/ml) or an equivalent volume of KRPG buffer as a control. On a separate plate, compounds were added back to the neutrophils that were washed off compounds, and cells were stimulated to ensure neutrophils' capacity to respond. Neutrophils incubated with each compound without washing were also stimulated for comparison.
  • FIG. 8 shows the results of the reversibility test. Comp II appears to have the greatest reversibility.
  • neutrophils isolated as described above were incubated with selected compound for 30 min and stimulated with FMLF (100 nM) or by contact with L. monocytogenes or S. typhimurium .
  • FMLF 100 nM
  • L. monocytogenes or S. typhimurium For the bacteria triggered samples, neutrophils were incubated with DMSO or a compound for 30 min at 37° C. and then exposed to 10% autologous serum-opsonized Salmonella typhimurium (ATCC 14028s) (a Gram-negative bacterium) or Listeria monocytogenes (ATCC 104035) (a Gram-positive bacterium), at a multiplicity of infection of 0.5 bacteria per neutrophil. H 2 O 2 release was measured as above.
  • FIG. 9A shows the effect of Comp II and Comp V on fMLF stimulated respiratory burst.
  • FIG. 9B shows the effect of Comp I, III, IV, and V on on fMLF stimulated respiratory burst in human neutrophils.
  • FIG. 10A shows the effect of Comp I and Comp V on bacteria-triggered respiratory burst.
  • the effect of Comp II-V on bacteria-triggered respiratory burst in neutrophils is shown in FIG. 10B .
  • H 2 O 2 release triggered by fMLF a bacteria-released chemoattractant, was blocked by all four active compounds.
  • neutrophils were treated with a compound of the present invention or DMSO and then activated with PMA or TNF.
  • Human neutrophils, isolated as described herein above were plated on FBS-coated glass coverslips and incubated or not with a compound of the present invention at 37° C. for 30 min, followed by stimulation with TNF (100 ng/ml), PMA (100 ng/ml) or an equal volume of KRPG, fixed, and photographed with phase-contrast microscope.
  • TNF 100 ng/ml
  • PMA 100 ng/ml
  • KRPG a compound of the present invention
  • Comp I treated neutrophils consistently showed very heterogeneous morphology upon TNF stimulation. They display almost round and close to non-stimulated cell morphology to quite advanced, yet arrested phenotype before full spreading.
  • Comp II and IV treated cells were arrested after flattening and extending several filopodia, as shown in FIG. 12A
  • Comp III treated cells after extending a few pseudopodia, as shown in FIG. 12B .
  • Comp III seems to block cell spreading at the earliest stage among these four compounds. None of the compounds impeded cell spreading induced by PMA. Control Comp V did not show any inhibition in cell spreading triggered by TNF nor PMA.
  • leukocytes For appropriate control of invading pathogens, leukocytes must migrate to the site(s) of infection. Failure to reach the site of infection can lead to devastating outcome for the host.
  • neutrophils were treated with a compound of the present invention and allowed them to transmigrate through the TNF activated human umbilical vein endothelial cell (HUVEC) layer.
  • HUVEC human umbilical vein endothelial cell
  • human umbilical vein endothelial cells were isolated by standard methods (Griesmacher et al., “17 Alpha-Ethinylestradiol Decreases Production and Release of Prostacyclin in Cultured Human Umbilical Vein Endothelial Cells,” Prostaglandins 38(4):431-8 (1989), which is hereby incorporated by reference in its entirety) and cultured in M199 medium (Gibco, Gaithersburg, Md.) supplemented with 20% normal human serum, penicillin, and streptomycin. Experiments were carried out on cells at passage two cultured on hydrated type I collagen gels in 96-well plates.
  • HUVEC were stimulated with TNF (50 pg/ml) in 5% CO 2 at 37° C. overnight (i.e., circa 12-18 hrs).
  • Neutrophils were isolated from the peripheral blood of healthy adult volunteers by density gradient sedimentation in a discontinuous gradient of Ficoll (Amersham Pharmacia, Piscataway, N.J.) and Histopaque (Sigma-Aldrich, St Louis, Mo.), washed in HBSS+0.1% HSA, resuspended to 0.5 ⁇ 10 6 cells/mL and added to HUVEC monolayers. Small molecule compounds were incubated with the PMNs at RT for 30 min before TEM and allowed to remain during the duration of the assay.
  • Transmigration data is expressed as the percentage of the total cells that transmigrated below the endothelial layer (% TEM). None of the compound appears to block either adhesion or transmigration of neutrophils through HUVECs, as shown in FIGS. 13A-B . Pretreatment of HUVECs with each compound before transmigration assay had no influence on the transmigration of non-treated neutrophils, indicating that none of these compounds interfere with TNF induced activation of HUVEC. Each of compounds I-IV blocked TNF elicited spreading of neutrophils, as shown in FIGS. 12A-B , yet did not interfere with the cytoskeletal rearrangement necessary for adhesion and transmigration through HUVEC. This suggests that none of these compounds are actually targeting components directly involved in cytoskeletal rearrangements. Rather, they might be interfering with the upstream events that can be bypassed when the signal is triggered through different receptors.
  • Tissue culture plates (Primaria, Falcon) were coated with 3 ml of FBS in 5% CO 2 at 37° C. for at least 1 h and washed twice with 0.9% saline. Isolated human neutrophils (1.5 ⁇ 10 6 ) were added to each plate containing 4 ml of reaction mixture, incubated with each compound at 37° C. for 30 min, and stimulated with either buffer control or TNF (100 ng/ml).
  • the cell lysate was passed through a 26-gauge needle six times to shear DNA and centrifuged at 20,000 ⁇ g for 15 min to remove cell debris and DNA.
  • the protein concentration was determined using a Bio-Rad (Indianapolis, Ind.) kit.
  • Cell lysates were separated by SDS-PAGE and transferred electrophoretically to nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, N.H.).
  • the membranes were incubated with 5% milk in TBST (1 M Tris-HCl, pH 7.5, 9% NaCl, 0.1% Tween-20) for 1 h at 37° C. and then overnight at 4° C.
  • FIG. 14A shows the effect on TNF triggered tyrosine phosphorylation of total cellular proteins.
  • FIG. 14B shows TNF triggered Src activity.
  • FIG. 14C shows TNF triggered Syk activity.
  • FIG. 14D shows TNF-induced phosphorylation of endogenous Pyk2 by Comp I-V.
  • tyrosine kinases such as Src, Syk, and Pyk2, which are shown to be critical in the signaling pathways triggered by TNF.
  • Kinase assays were performed according to the instructions provided by United Bioinformatica, Inc. ((UBI) Calgary, AB, Canada), from which recombinant Src and Syk were purchased.
  • Src kinase reaction buffer 100 mM Tris-HCl, pH 7.2, 125 mM MgCl 2 , 25 mM MnCl 2 , 2 mM EGTA, 0.25 mM NaOV 4 , 2 mM DTT
  • Src kinase substrate peptide 375 ⁇ M, UBI, Calgary, AB, Canada
  • [ ⁇ -32P]ATP was added to the enzyme.
  • TCA trichloroacetic acid
  • reaction buffer contained 50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 0.1 mM NaOV 4 , 0.15 M 2-mercaptoethanol and substrate was poly (Glu-4-Tyr)(4:1) (CSI Biointernational, France).
  • Glu-4-Tyr Glu-4-Tyr
  • Phox is a multi-subunit enzyme complex that generates H 2 O 2 upon activation. Activation of Phox requires an assembly of both membrane associated and cytosolic subunits at the plasma or phagosomal membrane as well as phosphorylation of several subunits.
  • the cytochrome b559 (also known as b558) components of phox (gp91 and gp22) are localized on the membranes of specific granules (Bjerrum et al, “Dual Granule Localization of the Dormant NADPH Oxidase and Cytochrome b559 in Human Neutrophils,” Eur J Haematol 43(1): 67-77 (1989), which is hereby incorporated by reference in its entirety), and recruitment of these subunits to the membrane occurs simultaneously with degranulation. This pathway is shown in FIG. 16 .
  • Comp II is not likely to block H 2 O 2 release by interfering with recruitment of the cytochrome b559 components of Phox, since degranulation takes place normally in Comp II treated cells.
  • Neutrophil lysates were prepared as above with 2.5% TritonX-100 lysis buffer (final concentration of 1% TritonX-100 after dilution in plate with residual KRPG). The cell lysate was passed through 26-gauge needle six times to shear DNA and centrifuged at 20,000 ⁇ g for 15 min to remove cell debris and DNA. The supernatant was centrifuged again at 100,000 ⁇ g for 1 h to pellet membrane fraction. The pellet was washed with PBS and resuspended in 2% TritonX-100 lysis buffer. Membrane fraction from each condition was separated by SDS-PAGE and western blotted with anti-phox 47 antibody. As shown in FIG.
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